The present invention relates generally to compositions for the prevention and treatment of tuberculosis. The invention is more particularly related to compositions comprising at least two Mycobacterium tuberculosis antigens, and the use of such compositions for treating and vaccinating against Mycobacterium tuberculosis infection.
Tuberculosis is a chronic, infectious disease, that is generally caused by infection with Mycobacterium tuberculosis. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If left untreated, serious complications and death typically result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance.
Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in AIDS patients, due to the depletion of CD4+ T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4+ T cells have been shown to be potent producers of gamma-interferon (IFN-xcex3), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice. While the role of IFN-xcex3 in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-xcex3 or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-xcex3 stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection. For a review of the immunology of M. tuberculosis infection see Chan and Kaufmann in Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, D.C., 1994.
Accordingly, there is a need in the art for improved compositions and methods for preventing and treating tuberculosis.
Briefly stated, this invention provides compositions and methods for preventing and treating M. tuberculosis infection. In one aspect, pharmaceutical compositions are provided that comprise a physiologically acceptable carrier and either (a) a first polypeptide and a second polypeptide, or (b) a fusion protein including a first polypeptide and a second polypeptide, wherein each of the polypeptides comprises an immunogenic portion of a M. tuberculosis antigen or a variant thereof. In specific embodiments, the first polypeptide comprises an immunogenic portion of a M. tuberculosis antigen having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 91, 107, 109, 111 and variants thereof, and the second polypeptide comprises an immunogenic portion of a M. tuberculosis antigen having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 79, 88, 115, 117, 118, 119 and variants thereof. In one preferred embodiment, the first polypeptide comprises an amino acid sequence of SEQ ID NO:107 and the second polypeptide comprises an amino acid sequence of SEQ ID NO:115. In another preferred embodiment, the first polypeptide comprises an amino acid sequence of SEQ ID NO:107 and the second polypeptide comprises an amino acid sequence of SEQ ID NO: 79.
Within other aspects, the present invention provides pharmaceutical compositions that comprise a physiologically acceptable carrier and either (a) a first DNA molecule and a second DNA molecule, or (b) a DNA fusion molecule comprising a first DNA molecule and a second DNA molecule, wherein each of the DNA molecules encodes an immunogenic portion of a M. tuberculosis antigen or a variant thereof. In specific embodiments, the first DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 33, 106, 108, 110 and variants thereof, and the second DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 46, 112, 116 and variants thereof. In one preferred embodiment, the first DNA molecule comprises a nucleotide sequence of SEQ ID NO:107 and the second DNA molecule comprises a nucleotide sequence of SEQ ID NO:115. In another preferred embodiment, the first DNA molecule comprises a nucleotide sequence of SEQ ID NO:107 and the second DNA molecule comprises a nucleotide sequence of SEQ ID NO: 17.
The invention also provides vaccines comprising an immune response enhancer and either (a) a first polypeptide and a second polypeptide, or (b) a fusion protein including a first polypeptide and a second polypeptide, wherein each of the polypeptides comprises an immunogenic portion of a M. tuberculosis antigen or a variant thereof. In specific embodiments, the first polypeptide comprises an immunogenic portion of a M. tuberculosis antigen having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 91, 107, 109, 111 and variants thereof, and the second polypeptide comprises an immunogenic portion of a M. tuberculosis antigen having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 79, 88, 115, 117, 118, 119 and variants thereof. In one preferred embodiment of this aspect of the present invention, the first polypeptide comprises an amino acid sequence of SEQ ID NO:107 and the second polypeptide comprises an amino acid sequence of SEQ ID NO: 115. In another preferred embodiment, the first polypeptide comprises an amino acid sequence of SEQ ID NO:107 and the second polypeptide comprises an amino acid sequence of SEQ ID NO: 79.
In a related aspect, the present invention provides vaccines comprising an immune response enhancer and either (a) a first DNA molecule and a second DNA molecule, or (b) a DNA fusion molecule comprising a first DNA molecule and a second DNA molecule, wherein each of the DNA molecules encodes an immunogenic portion of a M. tuberculosis antigen or a variant thereof. In specific embodiments, the first DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:33, 106, 108, 1 10 and variants thereof, and the second DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 46, 112, 116 and variants thereof. In one preferred embodiment, the first DNA molecule comprises a nucleotide sequence of SEQ ID NO:107 and the second DNA molecule comprises a nucleotide sequence of SEQ ID NO:115. In a further preferred embodiment, the first DNA molecule comprises a nucleotide sequence of SEQ ID NO: 107 and the second DNA molecule comprises a nucleotide sequence of SEQ ID NO: 17.
In preferred embodiments, the immune response enhancer employed in the inventive vaccines is an adjuvant. Most preferably, the adjuvant comprises 3-de-O-acylated monophosphoryl lipid A (3D-MPL) or the saponin QS21, or a combination of both 3D-MPL and QS21. The vaccines of the present invention may also, or alternatively, comprise an immunostimulatory cytokine or chemokine. Preferably, the vaccines are formulated in an oil in water emulsion.
In yet other aspects, methods are provided for inducing protective immunity in a patient, comprising administering to a patient an effective amount of one or more of the above pharmaceutical compositions or vaccines.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
FIGS. 1A and B illustrate the stimulation of proliferation and interferon-xcex3 production in T cells derived from a first and a second M. tuberculosis-immune donor, respectively, by the 14 Kd, 20 Kd and 26 Kd antigens described in Example 1.
FIG. 2 illustrates the stimulation of proliferation and interferon-y production in T cells derived from an M. tuberculosis-immune individual by the two representative polypeptides TbRa3 and TbRa9.
FIGS. 3A-D illustrate the reactivity of antisera raised against secretory M. tuberculosis proteins, the known M. tuberculosis antigen 85b and the inventive antigens Tb38-1 and TbH-9, respectively, with M. tuberculosis lysate (lane 2), M. tuberculosis secretory proteins (lane 3), recombinant Tb38-1 (lane 4), recombinant TbH-9 (lane 5) and recombinant 85b (lane 5).
FIG. 4A illustrates the stimulation of proliferation in a TbH-9-specific T cell clone by secretory M. tuberculosis proteins, recombinant TbH-9 and a control antigen, TbRa11.
FIG. 4B illustrates the stimulation of interferon-xcex3 production in a TbH-9-specific T cell clone by secretory M. tuberculosis proteins, PPD and recombinant TbH-9.
FIGS. 5A and B illustrate the stimulation of proliferation and interferon-xcex3 production in TbH9-specific T cells by the fusion protein TbH9-Tb38-1.
FIGS. 6A and B illustrate the stimulation of proliferation and interferon-xcex3 production in Tb38-1-specific T cells by the fusion protein TbH9-Tb38-1.
FIGS. 7A and B illustrate the stimulation of proliferation and interferon-xcex3 production in T cells previously shown to respond to both TbH-9 and Tb38-1 by the fusion protein TbH9-Tb38-1.
FIGS. 8A and B illustrate the stimulation of proliferation and interferon-xcex3 production in T cells derived from a first M. tuberculosis-immune individual by the representative polypeptides XP-1, RDIF6, RDIF8, RDIF10 and RDIF11.
FIGS. 9A and B illustrate the stimulation of proliferation and interferon-xcex3 production in T cells derived from a second M. tuberculosis-immune individual by the representative polypeptides XP-1, RDIF6, RDIF8, RDIF10 and RDIF11.
FIG. 10 illustrates the percentage survival of monkeys infected with M. tuberculosis following immunization with either saline, AS2 adjuvant alone, recombinant TbH9 (referred to as Mtb39) formulated in AS2 adjuvant, recombinant TbH9 plus recombinant TbRA35 (referred to as Mtb32) formulated in AS2, or BCG.
FIGS. 11A and B illustrate the bacteriological burden in the lungs and spleens, respectively, of guinea pigs infected with M. tuberculosis following immunization with either recombinant TbH9 (referred to as Mtb39) alone, recombinant TbRa35 (referred to as Mtb32) alone, or a combination of recombinant TbH9 and recombinant TbRa35.
FIG. 12 illustrates the bacteriological burden in the lungs of mice challenged with M. tuberculosis following immunization with either TbRa1 DNA alone, TbH9 DNA plus TbRa35 DNA, or a combination of TbH9 DNA, TbRa35 DNA and TbRa1 DNA.
SEQ. ID NO. 1 is the DNA sequence of TbRa1.
SEQ. ID NO. 2 is the DNA sequence of TbRa10.
SEQ. ID NO. 3 is the DNA sequence of TbRa11.
SEQ. ID NO. 4 is the DNA sequence of TbRa12.
SEQ. ID NO. 5 is the DNA sequence of TbRa13.
SEQ. ID NO. 6 is the DNA sequence of TbRa16.
SEQ. ID NO. 7 is the DNA sequence of TbRa17.
SEQ. ID NO. 8 is the DNA sequence of TbRa18.
SEQ. ID NO. 9 is the DNA sequence of TbRa19.
SEQ. ID NO. 10 is the DNA sequence of TbRa24.
SEQ. ID NO. 11 is the DNA sequence of TbRa26.
SEQ. ID NO. 12 is the DNA sequence of TbRa28.
SEQ. ID NO. 13 is the DNA sequence of TbRa29.
SEQ. ID NO. 14 is the DNA sequence of TbRa2A.
SEQ. ID NO. 15 is the DNA sequence of TbRa3.
SEQ. ID NO. 16 is the DNA sequence of TbRa32.
SEQ. ID NO. 17 is the DNA sequence of TbRa35.
SEQ. ID NO. 18 is the DNA sequence of TbRa36.
SEQ. ID NO. 19 is the DNA sequence of TbRa4.
SEQ. ID NO. 20 is the DNA sequence of TbRa9.
SEQ. ID NO. 21 is the DNA sequence of TbRaB.
SEQ. ID NO. 22 is the DNA sequence of TbRaC.
SEQ. ID NO. 23 is the DNA sequence of TbRaD.
SEQ. ID NO. 24 is the DNA sequence of YYWCPG.
SEQ. ID NO. 25 is the DNA sequence of AAMK.
SEQ. ID NO. 26 is the DNA sequence of TbL-23.
SEQ. ID NO. 27 is the DNA sequence of TbL-24.
SEQ. ID NO. 28 is the DNA sequence of TbL-25.
SEQ. ID NO. 29 is the DNA sequence of TbL-28.
SEQ. ID NO. 30 is the DNA sequence of TbL-29.
SEQ. ID NO. 31 is the DNA sequence of TbH-5.
SEQ. ID NO. 32 is the DNA sequence of TbH-8.
SEQ. ID NO. 33 is the DNA sequence of TbH-9.
SEQ. ID NO. 34 is the DNA sequence of TbM-1.
SEQ. ID NO. 35 is the DNA sequence of TbM-3.
SEQ. ID NO. 36 is the DNA sequence of TbM-6.
SEQ. ID NO. 37 is the DNA sequence of TbM-7.
SEQ. ID NO. 38 is the DNA sequence of TbM-9.
SEQ. ID NO. 39 is the DNA sequence of TbM-12.
SEQ. ID NO. 40 is the DNA sequence of TbM-13.
SEQ. ID NO. 41 is the DNA sequence of TbM-14.
SEQ. ID NO. 42 is the DNA sequence of TbM-15.
SEQ. ID NO. 43 is the DNA sequence of TbH-4.
SEQ. ID NO. 44 is the DNA sequence of TbH-4-FWD.
SEQ. ID NO. 45 is the DNA sequence of TbH-12.
SEQ. ID NO. 46 is the DNA sequence of Tb38-1.
SEQ. ID NO. 47 is the DNA sequence of Tb38-4.
SEQ. ID NO. 48 is the DNA sequence of TbHL-17.
SEQ. ID NO. 49 is the DNA sequence of TbL-20.
SEQ. ID NO. 50 is the DNA sequence of TbL-21.
SEQ. ID NO. 51 is the DNA sequence of TbH-16.
SEQ. ID NO. 52 is the DNA sequence of DPEP.
SEQ. ID NO. 53 is the deduced amino acid sequence of DPEP.
SEQ. ID NO. 54 is the protein sequence of DPV N-terminal Antigen.
SEQ. ID NO. 55 is the protein sequence of AVGS N-terminal Antigen.
SEQ. ID NO. 56 is the protein sequence of AAMK N-terminal Antigen.
SEQ. ID NO. 57 is the protein sequence of YYWC N-terminal Antigen.
SEQ. ID NO. 58 is the protein sequence of DIGS N-terminal Antigen.
SEQ. ID NO. 59 is the protein sequence of AEES N-terminal Antigen.
SEQ. ID NO. 60 is the protein sequence of DPEP N-terminal Antigen.
SEQ. ID NO. 61 is the protein sequence of APKT N-terminal Antigen.
SEQ. ID NO. 62 is the protein sequence of DPAS N-terminal Antigen.
SEQ. ID NO. 63 is the deduced amino acid sequence of TbRa1.
SEQ. ID NO. 64 is the deduced amino acid sequence of TbRa10.
SEQ. ID NO. 65 is the deduced amino acid sequence of TbRa11.
SEQ. ID NO. 66 is the deduced amino acid sequence of TbRa12.
SEQ. ID NO. 67 is the deduced amino acid sequence of TbRa13.
SEQ. ID NO. 68 is the deduced amino acid sequence of TbRa16.
SEQ. ID NO. 69 is the deduced amino acid sequence of TbRa17.
SEQ. ID NO. 70 is the deduced amino acid sequence of TbRa18.
SEQ. ID NO. 71 is the deduced amino acid sequence of TbRa19.
SEQ. ID NO. 72 is the deduced amino acid sequence of TbRa24.
SEQ. ID NO. 73 is the deduced amino acid sequence of TbRa26.
SEQ. ID NO. 74 is the deduced amino acid sequence of TbRa28.
SEQ. ID NO. 75 is the deduced amino acid sequence of ThRa29.
SEQ. ID NO. 76 is the deduced amino acid sequence of TbRa2A.
SEQ. ID NO. 77 is the deduced amino acid sequence of TbRa3.
SEQ. ID NO. 78 is the deduced amino acid sequence of TbRa32.
SEQ. ID NO. 79 is the deduced amino acid sequence of TbRa35.
SEQ. ID NO. 80 is the deduced amino acid sequence of TbRa36.
SEQ. ID NO. 81 is the deduced amino acid sequence of TbRa4.
SEQ. ID NO. 82 is the deduced amino acid sequence of TbRa9.
SEQ. ID NO. 83 is the deduced amino acid sequence of ThRaB.
SEQ. ID NO. 84 is the deduced amino acid sequence of TbRaC.
SEQ. ID NO. 85 is the deduced amino acid sequence of TbRaD.
SEQ. ID NO. 86 is the deduced amino acid sequence of YYWCPG.
SEQ. ID NO. 87 is the deduced amino acid sequence of TbAAMK.
SEQ. ID NO. 88 is the deduced amino acid sequence of Tb38-1.
SEQ. ID NO. 89 is the deduced amino acid sequence of TbH-4.
SEQ. ID NO. 90 is the deduced amino acid sequence of TbH-8.
SEQ. ID NO. 91 is the deduced amino acid sequence of TbH-9.
SEQ. ID NO. 92 is the deduced amino acid sequence of TbH-12.
SEQ. ID NO. 93 is the amino acid sequence of Tb38-1 Peptide 1.
SEQ. ID NO. 94 is the amino acid sequence of Tb38-1 Peptide 2.
SEQ. ID NO. 95 is the amino acid sequence of Tb38-1 Peptide 3.
SEQ. ID NO. 96 is the amino acid sequence of Tb38-1 Peptide 4.
SEQ. ID NO. 97 is the amino acid sequence of Tb38-1 Peptide 5.
SEQ. ID NO. 98 is the amino acid sequence of Tb38-1 Peptide 6.
SEQ. ID NO. 99 is the DNA sequence of DPAS.
SEQ. ID NO. 100 is the deduced amino acid sequence of DPAS.
SEQ. ID NO. 101 is the DNA sequence of DPV.
SEQ. ID NO. 102 is the deduced amino acid sequence of DPV.
SEQ. ID NO. 103 is the DNA sequence of ESAT-6.
SEQ. ID NO. 104 is the deduced amino acid sequence of ESAT-6.
SEQ. ID NO. 105 is the DNA sequence of TbH-8-2.
SEQ. ID NO. 106 is the DNA sequence of TbH-9FL.
SEQ. ID NO. 107 is the deduced amino acid sequence of TbH-9FL.
SEQ. ID NO. 108 is the DNA sequence of TbH-9-1.
SEQ. ID NO. 109 is the deduced amino acid sequence of TbH-9-1.
SEQ. ID NO. 108 is the DNA sequence of TbH-9-4.
SEQ. ID NO. 111 is the deduced amino acid sequence of TbH-9-4.
SEQ. ID NO. 112 is the DNA sequence of Tb38-1F2 IN.
SEQ. ID NO. 113 is the DNA sequence of Tb38-2F2 RP.
SEQ. ID NO. 114 is the deduced amino acid sequence of Tb37-FL.
SEQ. ID NO. 115 is the deduced amino acid sequence of Tb38-IN.
SEQ. ID NO. 116 is the DNA sequence of Tb38-1F3.
SEQ. ID NO. 117 is the deduced amino acid sequence of Tb38-1F3.
SEQ. ID NO. 118 is the DNA sequence of Tb38-1F5.
SEQ. ID NO. 119 is the DNA sequence of Tb38-1F6.
SEQ. ID NO. 120 is the deduced N-terminal amino acid sequence of DPV.
SEQ. ID NO. 121 is the deduced N-terminal amino acid sequence of AVGS.
SEQ. ID NO. 122 is the deduced N-terminal amino acid sequence of AAMK.
SEQ. ID NO. 123 is the deduced N-terminal amino acid sequence of YYWC.
SEQ. ID NO. 124 is the deduced N-terminal amino acid sequence of DIGS.
SEQ. ID NO. 125 is the deduced N-terminal amino acid sequence of AEES.
SEQ. ID NO. 126 is the deduced N-terminal amino acid sequence of DPEP.
SEQ. ID NO. 127 is the deduced N-terminal amino acid sequence of APKT.
SEQ. ID NO. 128 is the deduced amino acid sequence of DPAS.
SEQ. ID NO. 129 is the protein sequence of DPPD N-terminal Antigen.
SEQ ID NO. 130-133 are the protein sequences of four DPPD cyanogen bromide fragments.
SEQ ID NO. 134 is the N-terminal protein sequence of XDS antigen.
SEQ ID NO. 135 is the N-terminal protein sequence of AGD antigen.
SEQ ID NO. 136 is the N-terminal protein sequence of APE antigen.
SEQ ID NO. 137 is the N-terminal protein sequence of XYI antigen.
SEQ ID NO. 138 is the DNA sequence of TbH-29.
SEQ ID NO. 139 is the DNA sequence of TbH-30.
SEQ ID NO. 140 is the DNA sequence of TbH-32.
SEQ ID NO. 141 is the DNA sequence of TbH-33.
SEQ ID NO. 142 is the predicted amino acid sequence of TbH-29.
SEQ ID NO. 143 is the predicted amino acid sequence of TbH-30.
SEQ ID NO. 144 is the predicted amino acid sequence of TbH-32.
SEQ ID NO. 145 is the predicted amino acid sequence of TbH-33.
SEQ ID NO: 146-151 are PCR primers used in the preparation of a fusion protein containing TbRa3, 38 kD and Tb38-1.
SEQ ID NO: 152 is the DNA sequence of the fusion protein containing TbRa3, 38 kD and Tb38-1.
SEQ ID NO: 153 is the amino acid sequence of the fusion protein containing TbRa3, 38 kD and Tb38-1.
SEQ ID NO: 154 is the DNA sequence of the M. tuberculosis antigen 38 kD.
SEQ ID NO: 155 is the amino acid sequence of the M. tuberculosis antigen 38 kD.
SEQ ID NO: 156 is the DNA sequence of XP14.
SEQ ID NO: 157 is the DNA sequence of XP24.
SEQ ID NO: 158 is the DNA sequence of XP31.
SEQ ID NO: 159 is the 5xe2x80x2 DNA sequence of XP32.
SEQ ID NO: 160 is the 3xe2x80x2 DNA sequence of XP32.
SEQ ID NO: 161 is the predicted amino acid sequence of XP14.
SEQ ID NO: 162 is the predicted amino acid sequence encoded by the reverse complement of XP14.
SEQ ID NO: 163 is the DNA sequence of XP27.
SEQ ID NO: 164 is the DNA sequence of XP36.
SEQ ID NO: 165 is the 5xe2x80x2 DNA sequence of XP4.
SEQ ID NO: 166 is the 5xe2x80x2 DNA sequence of XP5.
SEQ ID NO: 167 is the 5xe2x80x2 DNA sequence of XP17.
SEQ ID NO: 168 is the 5xe2x80x2 DNA sequence of XP30.
SEQ ID NO: 169 is the 5xe2x80x2 DNA sequence of XP2.
SEQ ID NO: 170 is the 3xe2x80x2 DNA sequence of XP2.
SEQ ID NO: 171 is the 5xe2x80x2 DNA sequence of XP3.
SEQ ID NO: 172 is the 3xe2x80x2 DNA sequence of XP3.
SEQ ID NO: 173 is the 5xe2x80x2 DNA sequence of XP6.
SEQ ID NO: 174 is the 3xe2x80x2 DNA sequence of XP6.
SEQ ID NO: 175 is the 5xe2x80x2 DNA sequence of XP18.
SEQ ID NO: 176 is the 3xe2x80x2 DNA sequence of XP18.
SEQ ID NO: 177 is the 5xe2x80x2 DNA sequence of XP19.
SEQ ID NO: 178 is the 3xe2x80x2 DNA sequence of XP19.
SEQ ID NO: 179 is the 5xe2x80x2 DNA sequence of XP22.
SEQ ID NO: 180 is the 3xe2x80x2 DNA sequence of XP22.
SEQ ID NO: 181 is the 5xe2x80x2 DNA sequence of XP25.
SEQ ID NO: 182 is the 3xe2x80x2 DNA sequence of XP25.
SEQ ID NO: 183 is the full-length DNA sequence of TbH4-XP1.
SEQ ID NO: 184 is the predicted amino acid sequence of TbH4-XP1.
SEQ ID NO: 185 is the predicted amino acid sequence encoded by the reverse complement of TbH4-XP1.
SEQ ID NO: 186 is a first predicted amino acid sequence encoded by XP36.
SEQ ID NO: 187 is a second predicted amino acid sequence encoded by XP36.
SEQ ID NO: 188 is the predicted amino acid sequence encoded by the reverse complement of XP36.
SEQ ID NO: 189 is the DNA sequence of RDIF2.
SEQ ID NO: 190 is the DNA sequence of RDIF5.
SEQ ID NO: 191 is the DNA sequence of RDIF8.
SEQ ID NO: 192 is the DNA sequence of RDIF10.
SEQ ID NO: 193 is the DNA sequence of RDIF11.
SEQ ID NO: 194 is the predicted amino acid sequence of RDIF2.
SEQ ID NO: 195 is the predicted amino acid sequence of RDIF5.
SEQ ID NO: 196 is the predicted amino acid sequence of RDIF8.
SEQ ID NO: 197 is the predicted amino acid sequence of RDIF10.
SEQ ID NO: 198 is the predicted amino acid sequence of RDIF11.
SEQ ID NO: 199 is the 5xe2x80x2 DNA sequence of RDIF12.
SEQ ID NO: 200 is the 3xe2x80x2 DNA sequence of RDIF12.
SEQ ID NO: 201 is the DNA sequence of RDIF7.
SEQ ID NO: 202 is the predicted amino acid sequence of RDIF7.
SEQ ID NO: 203 is the DNA sequence of DIF2-1.
SEQ ID NO: 204 is the predicted amino acid sequence of DIF2-1.
SEQ ID NO: 205-212 are PCR primers used in the preparation of a fusion protein containing TbRa3, 38 kD, Tb38-1 and DPEP (hereinafter referred to as TbF-2).
SEQ ID NO: 213 is the DNA sequence of the fusion protein TbF-2.
SEQ ID NO: 214 is the amino acid sequence of the fusion protein TbF-2.
SEQ ID NO: 215 is the 5xe2x80x2 DNA sequence of MO-1.
SEQ ID NO: 216 is the 5xe2x80x2 DNA sequence for MO-2.
SEQ ID NO: 217 is the 5xe2x80x2 DNA sequence for MO-4.
SEQ ID NO: 218 is the 5xe2x80x2 DNA sequence for MO-8.
SEQ ID NO: 219 is the 5xe2x80x2 DNA sequence for MO-9.
SEQ ID NO: 220 is the 5xe2x80x2 DNA sequence for MO-26.
SEQ ID NO: 221 is the 5xe2x80x2 DNA sequence for MO-28.
SEQ ID NO: 222 is the 5xe2x80x2 DNA sequence for MO-29.
SEQ ID NO: 223 is the 5xe2x80x2 DNA sequence for MO-30.
SEQ ID NO: 224 is the 5xe2x80x2 DNA sequence for MO-34.
SEQ ID NO: 225 is the 5xe2x80x2 DNA sequence for MO-35.
SEQ ID NO: 226 is the predicted amino acid sequence for MO-1.
SEQ ID NO: 227 is the predicted amino acid sequence for MO-2.
SEQ ID NO: 228 is the predicted amino acid sequence for MO-4.
SEQ ID NO: 229 is the predicted amino acid sequence for MO-8.
SEQ ID NO: 230 is the predicted amino acid sequence for MO-9.
SEQ ID NO: 231 is the predicted amino acid sequence for MO-26.
SEQ ID NO: 232 is the predicted amino acid sequence for MO-28.
SEQ ID NO: 233 is the predicted amino acid sequence for MO-29.
SEQ ID NO: 234 is the predicted amino acid sequence for MO-30.
SEQ ID NO: 235 is the predicted amino acid sequence for MO-34.
SEQ ID NO: 236 is the predicted amino acid sequence for MO-35.
SEQ ID NO: 237 is the determined DNA sequence for MO-10.
SEQ ID NO: 238 is the predicted amino acid sequence for MO-10.
SEQ ID NO: 239 is the 3xe2x80x2 DNA sequence for MO-27.
SEQ ID NO: 240 is the full-length DNA sequence for DPPD.
SEQ ID NO: 241 is the predicted full-length amino acid sequence for DPPD.
As noted above, the present invention is generally directed to compositions and methods for the prevention and treatment of tuberculosis. In one aspect, the compositions of the subject invention include at least two isolated polypeptides that comprise an immunogenic portion of a M. tuberculosis antigen, or a variant of such an antigen. The inventive compositions may comprise a mixture of at least two isolated polypeptides or, alternatively, the polypeptides may be linked together to form a fusion protein. In a second aspect, the inventive compositions include at least two isolated DNA molecules, each DNA molecule encoding one or more of the above polypeptides. The isolated DNA molecules may be present within the composition as a mixture, or may be linked together to form a DNA fusion molecule.
As described in detail below, the inventors have determined that pharmaceutical compositions and/or vaccines comprising at least two of the above polypeptides or, alternatively, at least two DNA molecules encoding such polypeptides, are particularly efficacious in the induction of protective immunity against tuberculosis. In preferred embodiments, the inventive compositions comprise at least an immunogenic portion of the M. tuberculosis antigen TbH9, together with at least an immunogenic portion of either the M. tuberculosis antigen TbRa35 or the M. tuberculosis antigen Tb38.1. However, other combinations of the M. tuberculosis antigens described herein may also be effectively used in the treatment and prevention of tuberculosis, as may combinations of the inventive antigens with known M. tuberculosis antigens, such as the previously described 38 kD antigen (Andersen and Hansen, Infect. Immun. 57:2481-2488, 1989; Genbank Accession No. M30046).
As used herein, the term xe2x80x9cpolypeptidexe2x80x9d encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic portion of a M. tuberculosis antigen may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis antigen or may be heterologous, and such sequences may (but need not) be immunogenic. In general, the polypeptides disclosed herein are prepared in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure.
xe2x80x9cImmunogenic,xe2x80x9d as used herein, refers to the ability to elicit an immune response (e.g., cellular) in a patient, such as a human, and/or in a biological sample. In particular, antigens that are immunogenic (and immunogenic portions thereof) are capable of stimulating cell proliferation, interleukin-12 production and/or interferon-xcex3 production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M. tuberculosis-immune individual. Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247 and references cited therein. Such techniques include screening polypeptide portions of the native antigen for immunogenic properties. An immunogenic portion of a polypeptide is a portion that, within such assays, generates an immune response (e.g., proliferation, interferon-xcex3 production and/or interleukin-12 production) that is substantially similar to that generated by the full length antigen. In other words, an immunogenic portion of an antigen may generate at least about 20%, and preferably about 100%, of the proliferation induced by the full length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, and preferably about 100%, of the interferon-xcex3 and/or interleukin-12 induced by the full length antigen in the model assay described herein.
As used herein, the term xe2x80x9cDNA moleculexe2x80x9d includes both sense and anti-sense strands, and comprehends cDNA, genomic DNA, recombinant DNA and wholly or partially synthesized nucleic acid molecules.
The compositions and methods of the present invention also encompass variants of the above polypeptides and DNA molecules. A polypeptide xe2x80x9cvariant,xe2x80x9d as used herein, is a polypeptide that differs from the recited polypeptide only in conservative substitutions and/or modifications, such that the therapeutic, antigenic and/or immunogenic properties of the polypeptide are retained. A variant of a specific M. tuberculosis antigen will therefore stimulate cell proliferation and/or IFN-gamma in Th1 cells raised against that specific antigen. Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% homology to the identified polypeptides. For polypeptides with immunoreactive properties, variants may, alternatively, be identified by modifying the amino acid sequence of one of the above polypeptides, and evaluating the immunoreactivity of the modified polypeptide. Such modified sequences may be prepared and tested using, for example, the representative procedures described herein.
As used herein, a xe2x80x9cconservative substitutionxe2x80x9d is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
A nucleotide xe2x80x9cvariantxe2x80x9d is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA 2:183, 1983). Nucleotide variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology to the recited sequence. Such variant nucleotide sequences will generally hybridize to the recited nucleotide sequence under stringent conditions. As used herein, xe2x80x9cstringent conditionsxe2x80x9d refers to prewashing in a solution of 6xc3x97SSC, 0.2% SDS; hybridizing at 65xc2x0 C., 6xc3x97SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1xc3x97SSC, 0.1% SDS at 65xc2x0 C. and two washes of 30 minutes each in 0.2xc3x97SSC, 0.1% SDS at 65xc2x0 C.
In general, M. tuberculosis antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures, as described in detail below. Purified antigens may be evaluated for their ability to elicit an appropriate immune response (e.g., cellular) using, for example, the representative methods described herein. Immunogenic antigens may then be sequenced using techniques such as traditional Edman chemistry. See Edman and Berg, Eur. J. Biochem. 80:116-132, 1967.
Immunogenic antigens may also be produced recombinantly using a DNA sequence that encodes the antigen, which has been inserted into an expression vector and expressed in an appropriate host cell. DNA sequences encoding M. tuberculosis antigens may, for example, be identified by screening an appropriate M. tuberculosis genomic or cDNA expression library with sera obtained from patients infected with M. tuberculosis. Such screens may generally be performed using techniques well known to those of ordinary skill in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.
DNA sequences encoding M. tuberculosis antigens may also be obtained by screening an appropriate M. tuberculosis cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated antigens. Degenerate oligonucleotide sequences for use in such a screen may be designed and synthesized, and the screen may be performed as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and references cited therein). Polymerase chain reaction (PCR) may also be employed, using the above oligonucleotides in methods well known in the art, to isolate a nucleic acid probe from a cDNA or genomic library. The library screen may then be performed using the isolated probe.
Alternatively, genomic or cDNA libraries derived from M. tuberculosis may be screened directly using peripheral blood mononuclear cells (PBMCs) or T cell lines or clones derived from one or more M. tuberculosis-immune individuals. In general, PBMCs and/or T cells for use in such screens may be prepared as described below. Direct library screens may generally be performed by assaying pools of expressed recombinant proteins for the ability to induce proliferation and/or interferon-xcex3 production in T cells derived from an M. tuberculosis-immune individual.
Regardless of the method of preparation, the antigens (and immunogenic portions thereof) described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce proliferation and/or cytokine production (i.e., interferon-xcex3 and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from an M. tuberculosis-immune individual. The selection of cell type for use in evaluating an immunogenic response to a antigen will, of course, depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells and/or macrophages. An M. tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis (i.e., substantially free of disease symptoms). Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD) and an absence of any signs or symptoms of tuberculosis disease. T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods known to those of ordinary skill in the art. For example, a preparation of PBMCs (i.e., peripheral blood mononuclear cells) may be employed without further separation of component cells. PBMCs may generally be prepared, for example, using density centrifugation through Ficoll(trademark) (Winthrop Laboratories, NY). T cells for use in the assays described herein may also be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis-immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods known to those of ordinary skill in the art, to more accurately define individual T cell specificity. In general, antigens that test positive in assays for proliferation and/or cytokine production (i.e., interferon-xcex3 and/or interleukin-12 production) performed using T cells, NK cells, B cells and/or macrophages derived from an M. tuberculosis-immune individual are considered immunogenic. Such assays may be performed, for example, using the representative procedures described below. Immunogenic portions of such antigens may be identified using similar assays, and may be present within the polypeptides described herein.
The ability of a polypeptide (e.g., an immunogenic antigen, or a portion or other variant thereof) to induce cell proliferation is evaluated by contacting the cells (e.g., T cells and/or NK cells) with the polypeptide and measuring the proliferation of the cells. In general, the amount of polypeptide that is sufficient for evaluation of about 105 cells ranges from about 10 ng/mL to about 100 xcexcg/mL and preferably is about 10 xcexcg/mL. The incubation of polypeptide with cells is typically performed at 37xc2x0 C. for about six days. Following incubation with polypeptide, the cells are assayed for a proliferative response, which may be evaluated by methods known to those of ordinary skill in the art, such as exposing cells to a pulse of radiolabeled thymidine and measuring the incorporation of label into cellular DNA. In general, a polypeptide that results in at least a three fold increase in proliferation above background (i.e., the proliferation observed for cells cultured without polypeptide) is considered to be able to induce proliferation.
The ability of a polypeptide to stimulate the production of interferon-y and/or interleukin-12 in cells may be evaluated by contacting the cells with the polypeptide and measuring the level of interferon-xcex3 or interleukin-12 produced by the cells. In general, the amount of polypeptide that is sufficient for the evaluation of about 105 cells ranges from about 10 ng/mL to about 100 xcexcg/mL and preferably is about 10 xcexcg/mL. The polypeptide may, but need not, be immobilized on a solid support, such as a bead or a biodegradable microsphere, such as those described in U.S. Pat. Nos. 4,897,268 and 5,075,109. The incubation of polypeptide with the cells is typically performed at 37xc2x0 C. for about six days. Following incubation with polypeptide, the cells are assayed for interferon-xcex3 and/or interleukin-12 (or one or more subunits thereof), which may be evaluated by methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA) or, in the case of IL-12 P70 subunit, a bioassay such as an assay measuring proliferation of T cells. In general, a polypeptide that results in the production of at least 50 pg of interferon-xcex3 per mL of cultured supernatant (containing 104-105 T cells per mL) is considered able to stimulate the production of interferon-xcex3. A polypeptide that stimulates the production of at least 10 pg/mL of IL-12 P70 subunit, and/or at least 100 pg/mL of IL-12 P40 subunit, per 105 macrophages or B cells (or per 3xc3x97105 PBMC) is considered able to stimulate the production of IL-12.
In general, immunogenic antigens are those antigens that stimulate proliferation and/or cytokine production (i.e., interferon-xcex3 and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from at least about 25% of M. tuberculosis-immune individuals. Among these immunogenic antigens, polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed. In addition, antigens having superior therapeutic properties will not stimulate proliferation and/or cytokine production in vitro in cells derived from more than about 25% of individuals who are not M. tuberculosis-immune, thereby eliminating responses that are not specifically due to M. tuberculosis-responsive cells. Those antigens that induce a response in a high percentage of T cell, NK cell, B cell and/or macrophage preparations from M. tuberculosis-immune individuals (with a low incidence of responses in cell preparations from other individuals) have superior therapeutic properties.
Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use on experimental animals are described in detail below. Efficacy may be determined based on the ability of the antigen to provide at least about a 50% reduction in bacterial numbers and/or at least about a 40% decrease in mortality following experimental infection. Suitable experimental animals include mice, guinea pigs and primates.
Portions and other variants of M. tuberculosis antigens may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc., Foster City, Calif., and may be operated according to the manufacturer""s instructions. Variants of a native antigen may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
Recombinant polypeptides containing portions and/or variants of a native antigen may be readily prepared from a DNA sequence encoding the polypeptide using a variety of techniques well known to those of ordinary skill in the art. For example, supernatants from suitable host/vector systems which secrete recombinant protein into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant protein.
Any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
As noted above, in certain aspects the inventive compositions comprise fusion proteins or DNA fusion molecules. Each fusion protein comprises a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known M. tuberculosis antigen, such as the 38 kD antigen discussed above, together with variants of such fusion proteins. The fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides. The DNA fusion molecules of the present invention comprise a first and a second isolated DNA molecule, each isolated DNA molecule encoding either an inventive M. tuberculosis antigen or a known M. tuberculosis antigen.
A DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector, as described in detail below. The 3xe2x80x2 end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker, to the 5xe2x80x2 end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5xe2x80x2 to the DNA sequence encoding the first polypeptides. Similarly, stop codons require to end translation and transcription termination signals are only present 3xe2x80x2 to the DNA sequence encoding the second polypeptide.
The compositions of the present invention are preferably formulated as either pharmaceutical compositions or as vaccines for in the induction of protective immunity against tuberculosis in a patient. As used herein, a xe2x80x9cpatientxe2x80x9d refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, or may be free of detectable disease and/or infection. In other words, protective immunity may be induced to prevent or treat tuberculosis.
In one embodiment, pharmaceutical compositions of the present invention comprise at least two of the above polypeptides, either present as a mixture or in the form of a fusion protein, and a physiologically acceptable carrier. Similarly, vaccines comprise at least two of the above polypeptides and a non-specific immune response enhancer, such as an adjuvant or a liposome (into which the polypeptide is incorporated).
In another embodiment, a pharmaceutical composition and/or vaccine of the present invention may contain at least two DNA molecules, either present as a mixture or in the form of a DNA fusion molecule, each DNA molecule encoding a polypeptide as described above, such that the polypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be xe2x80x9cnaked,xe2x80x9d as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
A DNA vaccine and/or pharmaceutical composition as described above may be administered simultaneously with or sequentially to either a polypeptide of the present invention or a known M. tuberculosis antigen, such as the 38 kD antigen described above. For example, administration of DNA encoding a polypeptide of the present invention, either xe2x80x9cnakedxe2x80x9d or in a delivery system as described above, may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Any of a variety of adjuvants may be employed in the vaccines of this invention to enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are commercially available and include, for example, Freund""s Incomplete Adjuvant and Freund""s Complete Adjuvant (Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and quil A.
In the inventive vaccines, it is preferred that the adjuvant induces an immune response comprising Th1 aspects. Suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MLP) together with an aluminum salt. An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of 3D-MLP and the saponin QS21 as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739. Previous experiments have demonstrated a clear synergistic effect of combinations of 3D-MLP and QS21 in the induction of both humoral and Th1 type cellular immune responses. A particularly potent adjuvant formation involving QS21, 3D-MLP and tocopherol in an oil-in-water emulsion is described in WO 95/17210 and is a preferred formulation.
Routes and frequency of administration of the inventive pharmaceutical compositions and vaccines, as well as dosage, will vary from individual to individual and may parallel those currently being used in immunization using BCG. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), intralung, or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the patient from M. tuberculosis infection for at least 1-2 years. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 xcexcg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
The following Examples are offered by way of illustration and not by way of limitation.